Clinical trials of a new class of therapeutic agents: antisense oligonucleotides

Antisense oligodeoxynucleotides (ODNs) are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder whose genetic aetiology is well known. For this reason, researchers thought that if antisense drugs proved to be so specific there would be no side effects. However, toxicity-related problems arose in initial animal studies of antisense drugs in the early 1990s and since then companies have been using these compounds cautiously. In order to be useful therapeutically, an ODN must (a) exhibit reasonable stability in the physiological environment, (b) be taken up and retained in adequate quantities by the target cells, (c) specifically bind target mRNA with high affinity, (d) have an acceptable therapeutic ratio, free of unwanted toxic and non-specific side effects and (e) be easily synthesised in sufficient quantities to allow clinical use. Most of these criteria have already been met by ODNs recently used in this way. This review describes certain therapeutic applications of antisense techniques currently under investigation in oncology, haematopathology and inflammatory diseases.     

Antisense oligonucleotides as drugs for HIV treatment

Nowadays, several million people suffer from AIDS and more than 100 million people are forecasted to be infected with HIV. Among new drugs used to stop HIV virus infection, antisense oligodeoxynucleotides (ODNs) are under investigation and several biotechnology companies are currently developing antisense drugs. Antisense ODNs are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder. This review describes some of the clinical patents in the field of HIV treatments by antisense ODNs. These prior attempts at targeting HIV have largely focused on the nature of the chemical modification used in the ODN moiety. Although each of the described inventions have reported some degree of success in inhibiting some function of the virus, a general therapeutic scheme to target HIV has not yet been found.     

True antisense oligonucleotides with modified nucleotides restricted in the N-conformation

As first-generation antisense oligonucleotides, more than a dozen phosphorothioate oligodeoxynucleotides (PS ODNs) have been clinically developed, but only one has reached the market. To improve the drawbacks of PS ODNs, such as low affinity to target mRNA and non-specific binding to proteins, modified oligonucleotides with 2'-modified sugars such as 2'-O-(2-methoxy)ethyl and 2'-F modification or with bridged sugars such as oxyalkylene linkages between 2'-oxygen and 4'-carbon, have been synthesized as 2'-MOE, 2'-F RNA, 2',4'-BNA/LNA and ENA oligonucleotides. They have shown properties of higher affinity to complementary single-stranded RNA and DNA than those of PS ODNs due to their preorganized N-conformation. On the basis of the properties of these newly designed oligonucleotides, their in vitro and in vivo applications for gene silencing as true antisense oligonucleotides have been reported. In this review, antisense applications with these modified oligonucleotides are focused on.     

Antisense-inhibition of plasma membrane Ca2+ pump induces apoptosis in vascular smooth muscle cells

The effect of antisense oligodeoxynucleotides (ODNs) of plasma membrane Ca(2+)-pumping ATPase (PMCA) on rat aortic vascular smooth muscle cells (VSMCs) in primary culture was examined. More than 80% of the PMCA expressed in cultured VSMCs was the PMCA-1B subtype. Exposed to antisense ODNs against PMCA-1, not only the expression of the PMCA protein but also mRNA of PMCA-1B was diminished in a concentration-dependent manner. Extracellular Na(+)-independent (45)Ca(2+) efflux catalyzed via PMCA was inhibited with antisense ODNs. Both the resting and ionomycin- or ATP-stimulated levels of intracellular Ca(2+) were increased by antisense ODNs. Furthermore, prolonged treatment with antisense ODNs caused apoptosis in VSMCs. The occurrence of apoptosis was inhibited by FK506, a potent immunosuppressant. These results demonstrate that the PMCA was specifically inhibited by antisense ODNs and suggest that PMCA plays an important role in regulation of intracellular Ca(2+) concentrations, especially at the resting condition to prevent an occurrence of apoptosis that may be induced through the activation of calcineurin.     

Toxicology of antisense therapeutics

Targeting unique mRNA molecules using antisense approaches, based on sequence specificity of double-stranded nucleic acid interactions should, in theory, allow for design of drugs with high specificity for intended targets. Antisense-induced degradation or inhibition of translation of a target mRNA is potentially capable of inhibiting the expression of any target protein. In fact, a large number of proteins of widely varied character have been successfully downregulated using an assortment of antisense-based approaches. The most prevalent approach has been to use antisense oligonucleotides (ASOs), which have progressed through the preclinical development stages including pharmacokinetics and toxicological studies. A small number of ASOs are currently in human clinical trials. These trials have highlighted several toxicities that are attributable to the chemical structure of the ASOs, and not to the particular ASO or target mRNA sequence. These include mild thrombocytopenia and hyperglycemia, activation of the complement and coagulation cascades, and hypotension. Dose-limiting toxicities have been related to hepatocellular degeneration leading to decreased levels of albumin and cholesterol. Despite these toxicities, which are generally mild and readily treatable with available standard medications, the clinical trials have clearly shown that ASOs can be safely administered to patients. Alternative chemistries of ASOs are also being pursued by many investigators to improve specificity and antisense efficacy and to reduce toxicity. In the design of ASOs for anticancer therapeutics in particular, the goal is often to enhance the cytotoxicity of traditional drugs toward cancer cells or to reduce the toxicity to normal cells to improve the therapeutic index of existing clinically relevant cancer chemotherapy drugs. We predict that use of antisense ASOs in combination with small molecule therapeutics against the target protein encoded by the antisense-targeted mRNA, or an alternate target in the same or a connected biological pathway, will likely be the most beneficial application of this emerging class of therapeutic agent.     

Section Review: Biologicals & Immunologicals: Applications of antisense technology to both basic and clinical research

Antisense oligodeoxynucleotides (ODNs) are synthetic molecules typically designed to hybridise to the sense strand of the target gene messenger RNA (mRNA). The activity of these molecules reduces gene expression by disrupting the flow of information from gene to protein. The specificity of this interaction arises from the antisense ODN having a unique affinity for the primary sequence of the target gene due to nucleotide base complementarity. Specific inhibition of target gene products allows a rapidly increasing number of researchers to investigate gene function. Additionally, the reversible and specific nature of this interaction has initiated interest in antisense ODNs as a potentially novel class of therapeutics. Antisense molecules are already undergoing clinical trials as candidate therapeutics for several indications. Recent advances in our understanding of the activity of antisense ODNs, together with the advances in ODN chemistry, has made the notion of therapeutically active antisense molecules realistic. However, there remain many challenges in this field which must be overcome. Notably, the most commonly used chemical class of antisense ODNs, the phosphorothioates, has been associated with significant non-specific activities. This brief review aims to present in vivo applications of antisense in the domains of both basic and clinical research.     

Targeting of biotinylated oligonucleotides to prostate tumors with antibody-based delivery vehicles

Specific delivery of therapeutic agents to tumor sites remains a problem and requires a nontoxic carrier able to bind a specific tumor cell marker. Although antibodies have been utilized as protein carriers for different types of drugs, they have not been employed for delivery of antisense oligonucleotides (ODNs). In this study, we modified monoclonal antibodies to target biotinylated ODNs to prostatic tumors which express prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA). Modified antibodies retained immunoreactivity and demonstrated an ability to form complexes with ODNs. Biodistribution of (35)S-oligonucleotide-antibody complexes was also examined in nude mice bearing human derived LNCaP prostatic tumors. Delivering ODNs with modified prostate specific antibodies produced greater tissue uptake and higher blood levels with both PSA and PSMA directed antibodies. While tumor uptake was not significantly improved after 24 h, the overall results of this study provide some additional insights into the complexity of the antibody-mediated delivery of ODNs in vivo. The strategies employed here for the selective delivery of ODNs warrant further investigation.     

Inhibition of Ras p21 synthesis by antisense undecamers with uniform and specifically arranged phosphorothioate linkages

The design of chimeric oligodeoxynucleotides (ODNs) in which certain phosphodiester linkages are replaced by phosphorothioate (PS) aims to decrease non-sequence-specific effects of uniform PS ODNs and to preserve the PS-provided protection against exo- and endonucleases. This study has, for the fist time, directly compared the differences in nuclease resistance, cellular uptake, antisense potency and sequence specificity of PS and end-capped, pyrimidine-protected (PPS) undecamer ODNs, that are complementary to the initiation codon region of human Ha-ras mRNA. At concentrations above 5 microM, both PS and PPS undecamers were moderately and equally stable for over 48 h in complete medium with RS485 cells overexpressing Ha-ras. They were completely stable at 0.4 microM when complexed with Lipofectin reagent that enhanced cellular uptake up to 9-fold. Both the antisense PPS and PS undecamers produced well-defined inhibition of Ras p21 synthesis in both cell-free and cell-based assays. However, non-sequence-specific effects of the uniform phosphorothioates were still significant. In contrast, the antisense PPS undecamer, when delivered to RS485 cells with Lipofectin reagent, inhibits human Ras p21 synthesis by more than 90% at a concentration of 3.2 microM, while the effect of controls with inverted, mismatched or scrambled sequence was minimal (5% or less) on p21 synthesis and RS485 cell growth.     

定量构效关系方程对靶向蛋白激酶 C-α m RNA的反义药物的活性预测(英文)

以往研究对 34个针对蛋白激酶 C- α m RNA二级结构设计的反义药物 (AS- ODNs)进行了定量构效关系 (QSAR)分析并得到 QSAR方程 .本研究再次设计 1 0个 AS- ODNs验证 QSAR方程对药物活性预测的可靠性 .结果显示其中 4个 AS- ODNsIC50 值明显低于阳性对照 ISIS352 1 (P<0 .0 5) . 8个AS- ODNs与 ISIS352 1的实测 IC50 与 QSAR方程预测相符 ,实测与预测 IC50 之间相关系数为 0 .76(P<0 .0 5) .两个 AS- ODNs,AP1 2 61 (2 0 )和 AP0 1 86(2 0 )的实测 IC50 与预测不符 .结果提示 QSAR方程一定程度上反映了 AS- ODNs活性与靶结构的关系 ,对预测反义药物生物活性有益 .但 QSAR方程难以解释的其他因素需进一步研究以优化反义药物设计     

The use of antisense oligodeoxynucleotides (aODNs) for the therapy of cancer

Although specific cancer targets are difficult to identify, the recent development of antisense oligodeoxynucleotides (aODNs) as inhibitors of gene expression has been shown to provide a new and useful tool in antiblastic management. aODNs are able to specifically interact with gene or mRNA sequences and inhibit the expression of relevant molecules for cancer pathogenesis and progression. Since alpha-DNA polymerase (pol-alpha) plays an essential role in cell proliferation, aODNs to pol-alpha have been synthesized in order to block mRNA translation and affect the growth of MDA-MB 231, human breast cancer cell line and SW626 ovarian cancer cells. A rapid colorimetric test (MTT assay) which measures cell growth and survival has been employed to evaluate the effects induced by ODN treatment. The present experimental results demonstrate that the aODNs to pol-alpha are able to significantly affect cell proliferation. This study provides an encouraging basis for the exploitation of ODNs as therapeutic agents in vitro and in future clinical application.     

Targeting of biotinylated oligonucleotides to prostate tumors with antibody-based delivery vehicles

Specific delivery of therapeutic agents to tumor sites remains a problem and requires a nontoxic carrier able to bind a specific tumor cell marker. Although antibodies have been utilized as protein carriers for different types of drugs, they have not been employed for delivery of antisense oligonucleotides (ODNs). In this study, we modified monoclonal antibodies to target biotinylated ODNs to prostatic tumors which express prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA). Modified antibodies retained immunoreactivity and demonstrated an ability to form complexes with ODNs. Biodistribution of ³⁵S-oligonucleotide–antibody complexes was also examined in nude mice bearing human derived LNCaP prostatic tumors.

Delivering ODNs with modified prostate specific antibodies produced greater tissue uptake and higher blood levels with both PSA and PSMA directed antibodies. While tumor uptake was not significantly improved after 24 h, the overall results of this study provide some additional insights into the complexity of the antibody-mediated delivery of ODNs in vivo. The strategies employed here for the selective delivery of ODNs warrant further investigation.     

Drugs made of RNA: development and application of engineered RNAs for gene therapy

During the past decade, RNA has become a focus of investigation into new therapeutic schemes: antisense RNA, interfering RNA and trans-cleaving ribozymes are used to silence undesired gene expression. As an additional option with its own therapeutic potential, ribozymes may be employed to specifically alter the sequence of RNA. Among these RNA based strategies the mode of action varies: while antisense and interfering RNAs are capable of making specific contacts to other RNA molecules with the result of employing the cellular machinery for degradation of the RNA target, trans-cleaving ribozymes fold into specific three-dimensional structures to form catalytic centres and to specifically cleave a chosen RNA target. Beyond this, trans-splicing ribozymes have been engineered to first cleave a RNA target followed by ligation of a new RNA fragment delivered with the ribozyme. The latter strategy potentially extends the application of ribozymes from inhibition of gene expression to RNA repair, i. e. correction of genetic disorders at the level of RNA, and has already shown promising results in cell culture experiments. On the other side, advances in RNA synthesis, ribozyme engineering, delivery methods and expression systems have greatly enhanced the prospects of ribozymes, antisense and interfering RNAs in gene therapy. This review provides an overview of existing strategies for potential RNA based gene therapy. It is focussed on the engineering of ribozymes and functional RNAs to be used as drugs and on the basic molecular principles of their action.     

Novel non-endocytic delivery of antisense oligonucleotides

Antisense oligonucleotides (ONs) have several properties that make them attractive as therapeutic agents. Hybridization of antisense ONs to their complementary nucleic acid sequences by Watson-Crick base pairing is a highly selective and efficient process. Design of therapeutic antisense agents can be made more rationally as compared to most traditional drugs, i.e., they can be designed on the basis of target RNA sequences and their secondary structures. Despite these advantages, the design and use of antisense ONs as therapeutic agents are still faced with several obstacles. One major obstacle is their inefficient cellular uptake and poor accessibility to target sites. In this article, we will discuss key barriers affecting ON delivery and approaches to overcome these barriers. Current methods of ON delivery will be reviewed with an emphasis on novel non-endocytic methods of delivery. ONs are taken up by cells via an endocytic process. The process of ON release from endosomes is a very inefficient process and, hence, ONs end up being degraded in the endosomes. Thus, ONs do not reach their intended site of action in the cytoplasm or nucleus. Delivery systems ensuring a cytoplasmic delivery of ONs have the potential to increase the amount of ON reaching the target. Here, we shall examine various ON delivery methods that bypass the endosomal pathway. The advantages and disadvantages of these methods compared to other existing methods of ON delivery will be discussed.     

Combinatorial application of nucleic acid-based agents targeting protein kinases for cancer treatment

The progress made in cancer biology, genetics and biotechnology has led to a major transition in cancer drug design and development, from an emphasis on non-specific, cytotoxic agents to specific, molecular-targeted smart cancer drugs. Many of these targeted agents have shown to have improved selectivity for cancer versus normal cells and are associated with better anti-tumor efficacy and lower toxicity. The new generation of anti-cancer drugs requires low concentrations and minimizes unwanted side effects. Their use leads to enhanced anti-cancer effects and to a reduction of chemotherapy resistance. Still, resistance to common chemotherapeutic agents is a major obstacle in cancer treatment. Silencing of cancer-relevant genes is a challenging strategy to reduce resistance and to sensitize cancer cells towards anti-neoplastic agents. Resistance can be an intrinsic problem of the tumor or can be acquired during the life time of the tumor. A fascinating species of anti-cancer drugs include antisense oligonucleotides (ASOs) or small interfering RNAs (siRNAs) which are able to specifically down-regulate the expression of the target genes. The combination of nucleic acid-based agents with anti-neoplastic drugs can induce synergistic induction of cell cycle arrest, apoptosis and reduced cell proliferation in vitro or tumor growth in vivo. These two strategies (ASOs and siRNAs) will help to improve current therapeutic regimens. In addition, the combination of targeted drugs with common chemotherapeutic agents might be able to make resistant cells again sensitive towards a chemotherapeutic agent.